1. Field of the Invention
The present invention relates to an integral multilayer analytical element for use in measuring alkaline phosphatase activity in a liquid sample, and more particularly to an integral multilayer analytical element for use in measuring alkaline phosphatase activity useful for the analysis of aqueous liquid samples, particularly for clinical test using body fluids as samples.
2. Description of Prior Arts
It is very important to measure alkaline phosphatase activity present in the human body fluid in clinical test. Accordingly, the measurement of alkaline phosphatase (hereinafter, sometimes, referred to as ALP) activity in the body fluid has been made in the diagnoses of hepatopathy including obstructive jaundice and bone diseases including osteoncus.
Since a method for measuring alkaline phosphatase activity had been proposed by H. D. Kay [see, J. Biol. Chem., 88 235 (1930)], various methods have been studied and proposed. Particularly, the measurement had been efficiently simplified by a method developed by E. J. King [see, Biochem. J., 33 1185, 1939], wherein an aryl phosphate or the aryl phosphate, especially a self-developing p-nitrophenyl phosphate is used as the substrate.
The above-mentioned measuring method have been further studied by researchers. As a result, it has been found that the optimum pH in the color forming reaction is about 10, that the presence of a certain alcohol is preferred, and that an inorganic ion, particularly magnesium ion, is preferably present as an activator. Thus, there has been established IFCC method [see, Clin. Chimica, Acta., (1983) 339F-367F] wherein disodium p-nitrophenyl phosphate is used as a substrate.
However, this method has disadvantages in that the solution of disodium p-nitrophenyl phosphate must be prepared just at the time of measurement and is unstable. The time during which the solution can be preserved at 20.degree. to 26.degree. C. is only for 8 hours at most. Further, disodium p-nitrophenol phosphate is unstable even at freezer temperatures as pointed out by Amador. E., et al. [see, J. Amer. Med. Ass., 184 953 (1963)]. Accordingly, the potency of the reagent must be assayed before measurement and great care must be taken of the preservation of the reagent during the period of time from the preparation through measurement.
The problem in the instability of disodium p-nitrophenyl phosphate has been greatly improved by the method described in Japanese Patent Publication No. 45(1970)34827 wherein an organic amine salt of p-nitrophenyl phosphate are used. Further, an amine salt of thymolphthalein monophosphate which has an absorption spectrum in the region of wavelengths longer than that of p-nitrophenyl phosphate has been synthesized by John M. Ellickson, et al. [see, Clinical Chemistry, Vol. 19, No. 6, 1973, page 664]. The preservation of the substrates per se have been thus greatly improved, and it becomes possible that the substrate can be easily incorporated in a reagent kit for the measurement of ALP.
A rapid measurement system with a simple operation in clinical test is highly desired by medical persons such as medical doctors. Hence, methods for measuring ALP using integral multilayer analytical elements which are easy to handle have been developed and the improvements of the elements are being made.
An example of such integral multilayer analytical element comprises a water-absorbing layer containing a color forming reagent and a hydrophilic polymer binder and a porous spreading layer provided on a transparent support as reported by B. Walter [see, Analytical Chemistry, (1983) 55 498A]. Further, a number of other analytical elements have been known.
However, in order to perform the analysis of ALP using a dry analytical method, the degree of the improvement in the preservation of substrates such as p-nitrophenyl phosphate, etc. is still unsatisfactory. Therefore, it is highly desired to develop an integral multilayer analytical element for use in measuring alkaline phosphatase activity, which is free from the problem of insufficient preservation, particularly insufficient preservation of substrate.